The utility of fullerenes as biologically active compounds has given an impetus to intensive development of the chemistry of functionalized derivatives of fullerenes, especially following the discovery of a high antiviral activity in some water-soluble fullerene (see Partha, R., and Conycrs, J. L. “Biomedical Applications of Functionalized Fullerene-Based Nanomaterials.” Int. J. Nanomedicine, 2009 (4), 261-75; U.S. Pat. No. 6,204,391, 2005, “Water Soluble Fullerenes with Antiviral Activity;” R. Bakry et al., “Medicinal Application of Fullerenes,” International Journal of Nanomedicine, 2007 (4), 639-649; and Z. Zhu, D. 1. Schuster, and M. Tuckermann, “Molecular Dynamics Study of the Connection between Flap Closing and Binding of Fullerene-Based Inhibitors of the HIV-1 Protease”, Biochemistry, 2003, vol. 42, 1326-1333).
The medical use of fullerene derivatives is based on the lipophilic properties of the fullerene core, which enables fullerene derivatives to permeate cellular membranes, and the ability of fullerenes to generate in high quantum yield singlet oxygen, which splits DNAs. These properties endow functionalized fullerene derivatives with cytotoxic, antiviral, and other properties (see Bedrov, D., Smith, G. D., Davande, H., “Passive transport of fullerenes through a lipid membrane,” J. Phys. Chem., B, 2008, Vol. 112., pp. 2078-84; Qiao, R., and Roberts A. E., “Translocation of Fullerene and Its Derivatives across a Lipid Bilayer”, Nano Lett., 2007, Vol. 7, pp. 614-9; Nelsen, G. D., et al., “in vivo Biology and Toxicology of Fullerenes and their Derivatives”, Basic and Clinical Pharnnacology and Toxicology, 2008, Vol. 103, pp. 197-208).
Hydrated fullerene species have a high biological activity as bioantioxidants, which is due to the formation of active structural species of water clusters coordinated to the fullerene sphere (see Andrievsky, G. V., Brushkov, V. L, Tykhonov, A. A., and Gudkov S. V., “Peculiarities of the Antioxidant and Radioprotective Effects of Hydrated C60 Fullerene Nanostructures in vitro and in vivo.” Free Radical Biology and Medicine, 2009, vol. 47, pp. 786-793).
The main problem hampering biological studies of fullerenes and their derivatives and the creation of medicaments on their basis arises from the difficulty of solubilizing fullerene systems in aqueous solutions.
A promising method for preparing water-soluble fullerene compositions is to chemically modify the fullerene sphere with hydrophilic solubilizing ligands.
Currently, a wide range of functionalized fullerenes have been prepared, wherein hydrophilic moieties are present in the side chains of ligands attached to the fullerene (the detergent type of complex), as well as spherical derivatives wherein polar groups are distributed over the fullerene sphere (this type includes fullerenols and amino adducts).
Amino acid derivatives of fullerenes have the greatest potential for use.
Non-native amino acids of the aliphatic raw containing six or more of methylene groups have some specific features in the context of hydration and biochemical activity. Spectroscopic studies of water structure in aqueous solutions of amino acids show that increasing the number of methylene groups spacing the amino group and the carboxy group enhances the destruction of water clusters. Pharmacological studies of derivatives of the extensive series of R—(CH)nCOOH amino acids showed a higher activity in systems where n is equal to or is higher than six.
Spherical amino acid derivatives of fullerene C60 prepared by the reaction of nucleophilic addition of amino acids to the fullerene sphere at the amino group are described in Russian Federation patents Nos. 2196602, 2124022, and 2236852, and these patents can serve as the most pertinent pieces of prior art for our invention.
In the Russian Federation patent no. 2196602, there is claimed a method for inhibiting the reproduction of HIV and CMV infections by means of compounds based on amino acid and dipeptide fullerene derivatives. The amino acid fullerene derivatives used in that patent are sodium salts of fullerene aminocaproic acid and fullerene aminobutiric acid.
In the Russian Federation patent no. 2124022, in order to prepare fullerene aminocaproic acid, an aqueous solution of a potassium salt of aminocaproic acid and 18-crown-6 is added to a solution of fullerene in o-dichlorobenzene. The reaction mass is stirred for 6 to 8 hours at 60° C. Then, the solvents are distilled off, the residue is treated with a saturated potassium chloride solution, and the fullerene derivative residue is washed with water. The target product is obtained in quantitative yield. The resulting (monohydro)N-fullerene aminocaproic acid is soluble in dimethyl sulfoxide, dimethylformamide, and pyridine. The conditions for the final product to be separated are not defined in the synthesis method claimed in that patent.
The major drawback of the compounds prepared as described above, which are monoaddition products, consists in their water insolubility. One more drawback of the above-cited invention consists in that the phase-transfer catalyst used in the synthesis is crown ether, which is difficult to separate from the reaction products.
The Russian Federation patent no. 2236852 protects an agent for inhibiting the reproduction of enveloped viruses, this agent being fullerene polycarboxylic acid anions of general formula C60Hn[(CH2)mC(O)O−]n prepared by reacting the fullerene and an amino acid salt in an organic solvent medium in the presence of a poly(alkylene oxide).
In order to prepare those compounds, to a solution of fullerene in o-dichlorobenzene (or toluene, or another organic solvent), an amino acid is added as a salt (potassium or sodium salt) and then a solubilizing agent is added. The order in which the amino acid and solubilizing agent are added is unimportant; they can be added as a premixed complex. Useful solubilizing agents are various poly(alkylene oxides) (polyethylene glycols having molar weights from 150 to 400 or higher than 400 (for example, PEG-1500), as well as polyethylene glycols having free terminal groups, but also those with substituted terminal groups (for example, polyethylene glycol dimethyl ester having a molar weight of 500). In order to increase reaction rates, any strong reducing agent (an alkali metal) is added. The fullerene-to-amino acid ratio is increased by more than 50 times. Conversion to the desired pharmaceutically acceptable salt, especially to a sodium or potassium salt, is performed by treating the acid with a suitable base or by adding a salt of a weak volatile acid. In particular, a water-insoluble fullerene polycarboxylic acid is converted to a more preferable pharmaceutically acceptable, water soluble salt, for example to a sodium salt. Addition of a salt of a weak volatile acid is performed via treating the solution with an alkali metal salt of a weak volatile acid. Upon concentrating the solution by evaporation or freeze drying, the weak acid is removed and mixed fullerene polycarboxylic acids are recovered as mixtures of their alkali metal salts. The target product of that invention has a constant composition; the content of the major substance in the target product is as low as 87.8%.
The major drawbacks of the fullerene amino acid derivatives prepared by the method shown in the cited patent consist in that this method produces a mixture of fullerene carboxylate anions in the form of both salt and acid species. An individual compound cannot be prepared by the method described in the cited patent. Furthermore, the fullerene poly(amino acids) prepared by this prior-art method in the acid form are almost water insoluble. Attempts at preparing a stable pharmaceutical composition with fullerene polycarboxylic anions failed, because compounds are precipitating during storage. Fullerene poly(amino acids) influence leukopoiesis: they cause a shift of the leukocyte formula and induce the appearance of young forms of neutrophils (neutrophil metamyelocytes) in laboratory animals (rats and rabbits). In terms of safety (harmlessness), this indicates that these substances have toxicity which is responsible for the aforementioned alterations. The necessity of using in the synthesis of great excesses of a potassium or sodium salt of amino acids and great excesses of solvents gives rise to environmental problems in waste recycling, and increases the cost of the production process. For technological reasons alkali metals cannot be used to increase the reaction rate when chlorinated aromatic solvents are used.